Recombinant protein production is an essential activity for high throughput screening, functional validation, structural biology, and production of pharmaceutical polypeptides. Escherichia coli is a widely used organism for the expression of heterologous proteins because it easily grows to a high cell density on inexpensive substrates, and has well-established genetic techniques and expression vectors. However, this is not always sufficient for the efficient production of active biomolecules. In order to be biologically active, polypeptide chains have to fold into the correct native three-dimensional structure, including the appropriate formation of disulfide bonds, and may further require correct association of multiple chains.
Although the active state of the protein may be thermodynamically favored, the time-scale for folding can vary from milliseconds to days. Kinetic barriers are introduced, for example, by the need for alignment of subunits and sub-domains. And particularly with eukaryotic proteins, covalent reactions must take place for the correctly folded protein to form. The latter types of reaction include disulfide bond formation, cis/trans isomerization of the polypeptide chain around proline peptide bonds, preprotein processing and the ligation of prosthetic groups. These kinetic limitations can result in the accumulation of partially folded intermediates that contain exposed hydrophobic ‘sticky’ surfaces that promote self-association and formation of aggregates.
Recombinant synthesis of such complex proteins has had to rely on higher eukaryotic tissue culture-based systems for biologically active material. However, mammalian tissue culture based production systems are significantly more expensive and complicated than microbial fermentation methods. In addition, there continues to be questions regarding therapeutic products produced using materials derived from animal by-products.
As a eukaryote, P. pastoris pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression levels. As a yeast, it shares the advantages of molecular and genetic manipulations with Saccharomyces. These features make P. pastoris very useful as a protein expression system.
Many of the techniques developed for Saccharomyces may be applied to P. pastoris. These include transformation by complementation; gene disruption and gene replacement. In addition, the genetic nomenclature used for Saccharomyces has been applied to P. pastoris. There is also cross-complementation between gene products in both Saccharomyces and P. pastoris. Several wild-type genes from Saccharomyces complement comparable mutant genes in P. pastoris. 
Heterologous expression in P. pastoris pastoris can be either intracellular or secreted. Secretion requires the presence of a signal sequence on the expressed protein to target it to the secretory pathway. While several different secretion signal sequences have been used successfully, including the native secretion signal present on some heterologous proteins, success has been variable. A potential advantage to secretion of heterologous proteins is that P. pastoris pastoris secretes very low levels of native proteins. That, combined with the very low amount of protein in the minimal P. pastoris growth medium, means that the secreted heterologous protein comprises the vast majority of the total protein in the medium and simple removal of the yeast cells serves as the first step in purification of the protein.
Many species of yeast, including P. pastoris, are mating competent. This enables two distinct haploid strains to mate naturally and generate a diploid species possessing two complete sets of chromosomal copies.
Although P. pastoris has been used successfully for the production of various heterologous proteins, e.g., hepatitis B surface antigen (Cregg et al. (1987) Bio/Technology 5:479), lysozyme and invertase (Digan et al. (1988) Dev. Indust. Micro. 29:59; Tschopp et al. (1987) Bio/Technoloqy 5:1305), endeavors to produce other heterologous gene products in P. pastoris, especially by secretion, have given mixed results. At the present level of understanding of the P. pastoris expression system, it is unpredictable whether a given gene can be expressed to an appreciable level in this yeast or whether P. pastoris will tolerate the presence of the recombinant gene product in its cells. Further, it is especially difficult to foresee if a particular protein will be secreted by P. pastoris, and if it is, at what efficiency.
Various promoters have been derived from P. pastoris pastoris and used to regulate the expression of homologous and heterologous proteins in yeast. These promoters include in particular the alcohol oxidase promoters from the AOX1, AOX2 and mutant forms thereof as well as a promoter derived from the formaldehyde dehydrogenase gene FLD1. However, novel P. pastoris promoters especially P. pastoris promoters that are inducible or repressible under specific conditions and/or which provide for high expression yields in different yeast species are still needed.
The present invention satisfies this need and provides novel inducible promoters which are derived from P. pastoris pastoris and methods of use thereof to regulate the expression of structural genes operably linked thereto. In a preferred embodiment these promoters are used in the subject Assignee's proprietary improved methods and expression vectors that provide for the secretion of heterologous proteins, especially heteromultimers, from mating competent yeast, desirably polyploid yeast and most preferably diploid P. pastoris strains.